Calcium uptake inhibitors

ABSTRACT

This invention relates to 1-phenyl-3-phenyl-2-propyne-1-ones, the use of these compounds as calcium uptake inhibitors in leukocytes and thrombocytes, and pharmaceutical compositions containing these compounds as active ingredients, and the process of their preparation.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation of application Ser. No. 07/585,759, filed Sep.20, 1990 now abandoned.

FIELD OF THE INVENTION

This invention relates to 1-phenyl-3-phenyl-2-propyne-1-ones, the use ofthese compounds as calcium uptake inhibitors in leukocytes andthrombocytes, and pharmaceutical compositions containing these compoundsas active ingredients, and the process of their preparation.

BACKGROUND

Polymorphonuclear leukocytes (leukocytes) provide a principle means ofdefense against microbial infections. The response to invadingmicroorganisms causes activation of cellular oxidative processes(production of hydroxyl radicals) and nonoxidative processes (digestiveenzymes; myeloperoxidase, elastase, etc.) in order to effectively killthe microorganisms. However, the response of leukocytes to a foreignchallenge can also cause destruction to host tissues and play animportant part in the pathogenesis of a number of noninfectious diseaseconditions.

Leukocytes possess a wide variety of mechanisms that enable them torespond to foreign challenges which are initiated by cell surfacereceptors. Receptor activation or general cellular activation results inan altered cellular physiology causing the cell in itself to become"activated". The intracellular signaling molecules of activation areoften referred to as second messengers, the first messengers being theextracellar activating ligands themselves.

One of the major second messengers in many cells is the calcium ion(Ca⁺²). There are two general ways in which cell-surface receptors areknown to generate intracellular calcium signals. One is by activatingphospholipase C. This enzyme generates inositol trisphosphate which, inturn, releases stored calcium in the cell. Alternatively, cell receptorsmay open or close gated ion channels, letting calcium enter from outsidethe cell. Ca⁺² channels in the plasma membrane are of two types: (1)voltage sensitive calcium channels which are activated when a small andtransient flux of ions briefly alter the voltage across the plasmamembrane, or (2) receptor operated channels which are directly opened byreceptor ligands. The first mechanism operates mainly in voltagesensitive cells such as neurons and muscle cells. Many cells, likeleukocytes, are not primarily voltage sensitive cells but havecell-surface receptors that are functionally linked to receptorsensitive Ca⁺² channels in the plasma membrane. Binding of certainligands activates these receptors, thereby opening the channels andallowing Ca⁺² to enter the cytosol, where it then functions as a secondmessenger.

When cells are activated, corresponding to an influx of Ca⁺², structureswithin the cell that bind Ca⁺² are responsive to such changes, dependingon their relative affinity and specificity for calcium. A few Ca⁺²dependent proteins are known. The first such protein to be discoveredand characterized was troponin C found in electrically active skeletalmuscle cells. A later discovered calcium binding protein which isubiquitous in both voltage and receptor sensitive cells is calmodulin.Among the increasing number of cellular proteins known to be regulatedby calmodulin in a Ca⁺² dependent manner are some forms of cyclicnucleotide phosphodiesterase and adenylate cyclase, as well as membranebound calcium dependent ATPases, phosphorylase kinase, myosin lightchain kinases, and their association with the spindles of the mitoticapparatus and the bundles of actin filaments. Although the total numberof proteins that are calcium dependent or are affected by Ca⁺² dependentenzymes is not known it is clear that calcium is a requirement as ameans of activating these processes.

When leukocytes are activated, a number of events can occur which areimportant in leading to intracellular calcium mediated disease states.For example leukocytes, primarily the neutrophils, are thought to playan integral part in the symptoms and tissue injury of the host in thefollowing diseases; gout, rheumatoid arthritis, immune vasculitis,glomerulonephritis, inflammatory bowel disease, adult respiratorydistress syndrome, emphysema, asthma, thermal injury associated withhemolysis, and malignant neoplasms at sites of chronic inflammation(Malech and Gallin; 1987). It therefore appears desirable to inhibitCa⁺² uptake in leukocytes in order to alleviate or slow the progressionof these immune and inflammatory diseases associated with calciumuptake.

Ca⁺² uptake in leukocytes and amelioration of immune and inflammatorydiseases may also be extended to those diseases associated with the skinand dermal tissues. Included in the list of those topically relatedinflammatory diseases are those associated with skin and dermis,including neutrophil dermatoses, chronic dermatitis, psoriasis, contactdermatitis, atopic and seborrheic dermatitis, and acne.

Calcium is also an important mediator in thrombocytes (platelets) whereit is well known that Ca⁺² is a required mediator in the intrinsicpathway of blood coagulation. For example, the Ca⁺² requirement in theblood clotting process and thrombus formation is well known and theseprocesses can be inhibited in vitro, and to some degree in vivo, ifchelating agents such as EDTA, citrate, or oxalate are added to bindCa⁺². It is recognized that Ca⁺² plays an integral part of thefibrinolytic cascade and is an active mechanism by which thefibrinolytic response can be modulated therapeutically.

One of the primary functions of platelets occurs at a site of vascularinjury wherein they form clot aggregates to close the wound. Thisresponse can also have a number of detrimental side effects, for exampleduring ischemic reperfusion the thrombus formation can lead to occlusionof the artery, to the extent of causing a myocardial infarction. It istherefore desirable in many instances to inhibit Ca⁺² uptake inplatelets in order to control the thrombolytic response.

Ca⁺² entry into the cytosol, by various forms of receptor-mediatedactivation or by discharge of intracellular stores, is critically linkedto certain cellular events of leukocyte activation and plateletaggregation. Altering pathways of Ca⁺² mobilization provides a mechanismto modulate the responses of leukocytes and platelets. Thereforecompounds that inhibit Ca⁺² mobilization might be expected to reduceCa⁺² dependent disease processes associated with leukocyte activationand inhibition in immune and inflammatory diseases and in problemsinvolving platelet aggregation in certain thrombolytic conditions.

SUMMARY OF THE INVENTION

The present invention relates to novel 1-phenyl-3-phenyl-2-propyne-1-onederivatives which are useful as inhibitors of calcium uptake in (1)leukocytes associated with acute and chronic inflammatory and immunediseases, including related (2) disease of the skin and dermal tissues.Finally, this invention relates also to the use of1-phenyl-3-phenyl-2-propyne-1-one derivatives in the treatment ofcalcium dependent (3) processes of the thrombolytic system.

This inventions relates to compounds of the formula: ##STR1## whereinR₁, R₂ and R₃ each time taken are independently hydrogen; C₁ -C₆ alkyl;C₁ -C₆ alkoxy; halogen; --N(Y₁)(Y₂), wherein Y₁ and Y₂ are eachindependently hydrogen or C₁ -C₆ alkyl; or X_(z) --(Ar)--(CH₂)_(n)--O--, wherein Ar is phenyl or napthyl, n=0 or 1 X₂ =hydrogen, C₁ -C₆alkoxy or --N(Y₁).(Y₂), wherein Y₁ and Y₂ are as previously defined andZ=0, 1, 2;

or a pharmaceutically acceptable salt thereof.

The present invention also provides a method of inhibiting calciumuptake in a patient in need thereof comprising administration of atherapeutically effective inhibitory amount of a compound of formula(1).

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts the effect on intracellular calcium levels utilizing acompound of the invention.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "C₁ -C₆ alkyl" refers to a saturated straightor branched chain hydrocarbyl radical of one one to six carbon atoms.Included within the scope of this term are methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, n-hexyl, andthe like. The term "C₁ -C₆ alkoxy" refers to methoxy, ethoxy, propoxyand the like. It is also understood that the term X_(z)--(Ar)--(CH₂)_(n) --O-- specifically includes benzyloxy,(4-methoxy)benzyloxy, (4-dimethylamino)benzyloxy, and the like. The term"halogen" refers to a fluorine, chlorine, bromine, or iodine atom.

The compounds of formula (1) can be prepared by utilizing procedures andtechniques well known and appreciated by one of ordinary skill in theart. A general synthetic scheme for preparing compounds of formula (1)is set forth in Scheme A, wherein all substituents, unless otherwiseindicated, are previously defined. ##STR2##

In general, a 1-phenyl-3-phenyl-2-propyne-1-one compound of structure 5can be prepared according to Scheme A is a 3-step process.

In step a, the appropriate 2',2'-dibromostyrene compound of structure 2can be prepared by reacting the appropriate benzaldehyde compound ofstructure 1 with carbon tetrabromide and triphenylphosphine in asuitable aprotic solvent, such as methylene chloride.

Ins step b, the appropriate phenylacetylene compound of structure 3 canbe prepared by reacting the appropriate 2',2'-dibromostyrene compound ofstructure 2 with a non-nucleophilic base, such a n-butyllithium, in asuitable aprotic solvent, such as tetrahydrofuran.

In step c, the appropriate 1-phenyl-3-phenyl-2-propyne-1-one compound ofstructure 5 can be prepared by first reacting the appropriatephenylacetylene compound of structure 3 with a non-nucleophilic base,such as n-butyllithium, lithium hexamethyldisilazane, or lithiumdiisopropylamide, in a suitable aprotic solvent, such astetrahydrofuran. The corresponding lithium acetylide can then be reactedwith the appropriate N-methoxy-N-methylbenzamide (Weinreb reagent) ofstructure 4 to give the corresponding 1-phenyl-3-phenyl-2-propyne-1-onecompound of structure 5.

Alternatively, the appropriate 1-phenyl-3-phenyl-2-propyne-1-onecompound of structure 5 can be prepared by combining step b and step c.For example, the appropriate lithium acetylide compound obtained fromstep b can be used directly in the reaction with the appropriateN-methoxy-N-methylbenzamide (Weinreb reagent) of structure 4 to give thecorresponding 1-phenyl-3-phenyl-2-propyne-1-one compound of structure 5.

Starting materials for use in the general synthetic procedure outlinedin Scheme A are readily available to one of ordinary skill in the art.For example, certain N-methoxy-N-methylbenzamides are described inTetrahedron Letters, 22(39), 3815-18 (1981).

The following examples present typical syntheses as described in SchemeA. These examples are understood to be illustrative only and are notintended to limit the scope of the present invention in any way. As usedherein, the following terms have the indicated meanings: "g" refers tograms; "mmol" refers to millimoles; "mol" refers to moles; "mL" refersto milliliters; "bp" refers to boiling point; "°C." refers to degreesCelsius; "mm Hg" refers to millimeters of mercury; "mp" refers tomelting point; "mg" refers to milligrams; "μM" refers to micromolar;"μg" refers to micrograms.

EXAMPLE 1 1-Phenyl-3-(3,4,5-trimethoxyphenyl)-2-propyne-1-one Step a:3,4,5-Trimethoxy-2',2'-dibromostryene

Mix carbon tetrabromide (3.32 g, 10 mmol) and methylene chloride (7 mL)and cool to 0° C. Add, by dropwise addition, a solution oftriphenylphosphine (5.24 g, 20 mmol) in methylene chloride (7 mL) andstir at 0° C. for 30 minutes. Add, by dropwise addition,3,4,5-trimethoxybenzaldehyde (0.981 g, 5 mmol) in methylene chloride (10mL). Remove the ice bath and stir at room temperature for 30 minutes.Pour onto silica gel and elute with 5% ethyl acetate/hexane. Evaporatethe solvent in vacuo to yield 1.57 g of the title compound.

Step b and Step c: 1-Phenyl-3-(3,4,5-trimethoxyphenyl)-2-propyne-1-one

Place 3,4,5-trimethoxy-2',2'-dibromostryene (1.57 g, 4.46 mmol) andtetrahydrofuran (15 mL) under argon atmosphere and cool to -78° C. Add,by dropwise addition, n-butyllithium (5.85 mL of a 1.6M solution inhexane, 9.36 mmol) and stir for 1 hour at -78° C. Remove the coolingbath and stir for 1 hour at room temperature. Cool to -78° C. and addN-methoxy-N-methylbenzamide (1 mL, 6.6 mmol). Remove the cooling bathand stir at room temperature for 45 minutes. Partition between ethylether and water, separate the organic phase, dry, and evaporate thesolvent in vacuo to yield a solid. Recrystallize (1:2 chloroform/hexane)to yield 0.48 g of the title compound; mp 158°-60° C.

EXAMPLE 2 1-Phenyl-3-[3-(4-methoxybenzyloxy)phenyl]-2-propyne-1-one Stepa: 3-(4-Methoxybenzyloxy)-2',2'-dibromostyrene

Mix 3-hydroxybenzaldehyde (2.69 g, 22 mmol), p-methoxybenzyl chloride(2.71 mL, 20 mmol), potassium carbonate (3.04 g, 22 mmol), sodium iodide(1.5 g, 10 mmol) and acetone (60 mL). Heat at reflux for 16 hours, coolto room temperature and remove the solvent in vacuo to give a solidresidue. Partition the solid between ethyl ether and 6% sodiumhydroxide. Separate the organic phase and filter off any undissolvedsolid from the organic phase. Dry (MgSO₄), filter and evaporate thesolvent in vacuo to yield a solid. Slurry the solid with hexane, filterand air dry to yield 4.10 g (84.6%) of3-(4-methoxybenzyloxy)benzaldedyde; mp 78°-81° C.

Mix carbon tetrabromide (10.61 g, 32 mmol) and methylene chloride (15mL), place under argon atmosphere and cool to 0° C. Add, by dropwiseaddition, a solution of triphenylphosphine (16.77 g, 64 mmol) inmethylene chloride (15 mL) and stir at 0° C. for 30 minutes. Add, bydropwise addition, 3-(4-methoxybenzyloxy)benzaldedyde (3.88 g, 16 mmol)in methylene chloride (15 mL). Remove the ice bath and stir at roomtemperature for 1 hour. Pour onto silica gel and elute with 5% ethylacetate/hexane. Evaporate the solvent in vacuo to yield a solid. Slurrythe solid with hexane, filter and air dry to yield 3.8 g (1st crop) and0.405 g (2nd crop) of the title compound; mp 76°-9° C.

Step b: 3-(4-Methoxybenzyloxy)phenylacetylene

Place n-butyllithium (9 mL of a 2.5M solution in hexane, 22.5 mmol) andtetrahydrofuran (50 mL) under an argon atmosphere and cool in adry-ice/acetone bath. Add a solution of3-(4-methoxybenzyloxy)-2',2'-dibromostyrene (4.25 g, 10.7 mmol) intetrahydrofuran and stir for 1 hour at low temperature. Remove thecooling bath and stir for 1 hour at room temperature. Pour the yellowsolution into saturated aqueous ammonium chloride and extract into ethylether. Separate the organic phase, dry (MgSO₄), filter and evaporate thesolvent in vacuo to yield a solid. Recrystallize (hexane/chloroform) toyield 1.73 g of the title compound; mp 92°-5° C.

Step c: 1-Phenyl-3-[3-(4-methoxybenzyloxy)phenyl]-2-propyne-1-one

Place lithium hexamethyldisilazane (2 mL of a 1M solution intetrahydrofuran, 2 mmol) and tetrahydrofuran (8 mL) under an argonatmosphere and cool to 0° C. Add a solution of3-(4-methoxybenzyloxy)phenylacetylene (0.48 g, 2 mmol) intetrahydrofuran and stir the brown solution for 45 minutes at 0° C. AddN-methoxy-N-methylbenzamide (0.4 g, 2.4 mmol), remove the cooling bathand stir at room temperature for 1 hour. Partition the mixture betweenethyl ether and water, separate the organic phase and dry (MgSO₄).Filter and evaporate the solvent in vacuo to yield an oil. Filterthrough silica gel and elute with 10% ethyl acetate/hexane to yield acrude solid. Recrystallize (hexane, ethyl ether) to yield 0.26 g (1stcrop) and 0.15 g (2nd crop) of the title compound; mp 79°-80° C.

EXAMPLE 3 1-Phenyl-3-[(4-benzyloxy)phenyl]-2-propyne-1-one Step a: 4-Benzyloxy-2',2'-dibromostyrene

Mix carbon tetrabromide (3.32 g, 10 mmol) and methylene chloride (7 mL),place under argon atmosphere and cool to 0° C. Add, by dropwiseaddition, a solution of triphenylphosphine (5.24 g, 20 mmol) inmethylene chloride (7 mL) and stir at 0° C. for 30 minutes. Add, bydropwise addition, 4-benzyloxybenzaldehyde (1.06 g, 5 mmol) in methylenechloride (10 mL). Remove the ice bath and stir at room temperature for 2hours. Pour onto silica gel and elute with 5% ethyl acetate/hexane.Evaporate the solvent in vacuo to yield 1.68 g (91%) of the titlecompound.

Steps b and c: 1-Phenyl-3-[(4-benzyloxy)phenyl]-2-propyne-1-one

Place 4-benzyloxy-2',2-dibromostyrene (1.68 g, 4.56 mmol) andtetrahydrofuran (15 mL) under an argon atmosphere and cool to -78° C.Add, by dropwise addition, n-butyllithium and stir for 45 minutes at-78° C. Remove the cooling bath and stir for 1 hour at room temperature.Cool the yellow solution to -78° C. and add N-methoxy-N-methylbenzamide(1 mL, 6.6 mmol). Remove the cooling bath and stir at room temperatureovernight. Partition between ethyl ether and water, separate the organicphase and dry (MgSO₄). Filter and evaporate the solvent in vacuo toyield a solid. Recrystallize (hexane/chloroform) to yield 0.76 g of thetitle compound; mp 122°-124° C.

EXAMPLE 4 1-(3,4,5-Trimethoxyphenyl)-3-phenyl-2-propyne-1-one

Dissolve 3,4,5-trimethoxybenzoyl chloride (2.3 g, 10 mmol) andN,O-dimethylhydroxylamine hydrochloride (1.10 g, 11 mmol) inethanol-free chloroform at room temperature. Cool the solution to 0° C.and add pyridine (1.85 g, 22 mmol). Stir at ambient temperature for 1hour and evaporate the solvent in vacuo. Partition the residue betweenaqueous sodium chloride and a 1:1 mixture of ethyl ether and methylenechloride. Separate the organic phase, dry (Na₂ SO₄), and evaporate thesolvent in vacuo to yield an oil. Filter the oil through silica geleluting with 65% ethyl acetate/hexane to yield 2.5 g ofN-methoxy-N-methyl-(3,4,5-trimethoxy)benzamide.

Place lithium hexamethyldisilazane (3 mL of a 1M solution intetrahydrofuran, 3 mmol) and tetrahydrofuran (10 mL) under an argonatmosphere and cool to 0° C. Add phenylacetylene (0.33 mL, 3 mmol) andstir at 0° C. for 30 minutes. AddN-methoxy-N-methyl-(3,4,5-trimethoxy)benzamide (0.76 g, 3 mmol), removethe cooling bath and stir at room temperature for 1 hour. Partitionbetween ethyl ether and saturated aqueous sodium chloride, separate theorganic phase and dry (MgSO₄). Filter and evaporate the solvent invacuo. Pour onto silica gel and elute with 20% ethyl acetate/hexane toyield a crude solid. Recrystallize (hexane/ethyl acetate) to yield 0.31g of the title compound; mp 94°-96° C.

EXAMPLE 5 1-Phenyl-3-phenyl-2-propyne-1-one

Place lithium diisopropylamide (10 mL of a 1.5M solution in cyclohexane)and tetrahydrofuran (40 mL) under an argon atmosphere and cool to 0° C.Add, by dropwise addition, phenylacetylene (1.65 mL, 15 mmol). Removethe cooling bath and stir for 30 minutes as room temperature. Cool theyellow solution to 0° C. and add, by dropwise addition,N-methoxy-N-methylbenzamide (3.04 mL, 20 mmol). Remove the cooling bathand stir for 30 minutes at room temperature. Pour onto ethyl ether andwater, separate the organic phase and dry (MgSO₄). Filter and evaporatethe solvent in vacuo to yield an oil. Purify by silica gelchromatography (2% ethyl acetate/hexane) then purify further bydistillation (2×) to yield 1.9 g of the title compound; bp 210° C. invacuo.

EXAMPLE 6 1-Phenyl-3-(3-benzyloxyphenyl)-2-propyne-1-one Step a:3-Benzyloxy-2',2'-dibromostyrene

Place carbon tetrabromide (16.6 g, 50 mmol) and methylene chloride (25mL) under an argon atmosphere and cool to 0° C. Add, by dropwiseaddition, a solution of triphenylphosphine (26.2 g, 0.1 mol) inmethylene chloride (25 mL) and stir at 0° C. for 30 minutes. Add, bydropwise addition, a solution of 3-benzyloxybenzaldehyde (5.31 g, 25mmol) in methylene chloride (50 mL). Remove the cooling bath and stir atroom temperature for 1 hour. Pour onto silica gel (600 mL) and elutewith 5% ethyl acetate/hexane to yield 7.4 g, (80.4%) of the titlecompound.

Step b: 3-Benzyloxyphenylacetylene

Place 3-benzyloxy-2',2'-dibromostyrene (8.2 g, 22.3 mmol) andtetrahydrofuran (70 mL) under argon atmosphere and cool to -70° C. Add,by dropwise addition, n-butyllithium (18.7 mL of a 1.5M solution inhexane, 47 mmol). Stir at -70° C. for 1 hour and then for 1 hour at roomtemperature. Pour onto ethyl ether and water, separate the organic phaseand dry (MgSO₄). Filter and evaporate the solvent in vacuo. Purify bypreparative liquid chromatography (2% ethyl acetate/hexane) to yield2.73 g of the title compound.

Step c: 1-Phenyl-3-(3-benzyloxyphenyl)-2-propyne-1-one

Place lithium hexamethyldisilazane (15 mL of a 1M solution intetrahydrofuran, 15 mmol) and tetrahydrofuran (75 mL) under an argonatmosphere and cool with an ice-water bath. Add, by dropwise addition, asolution of 3-benzyloxyphenylacetylene (3.13 g, 15 mmol) intetrahydrofuran. Stir at 0° C. for 45 minutes, then add, by dropwiseaddition, N-methoxy-N-methylbenzamide (2.45 mL, 16 mmol). Remove thecooling bath and stir at room temperature for 1 hour. Partition betweenethyl ether and water, separate the organic phase and dry (MgSO₄).Filter and evaporate the solvent in vacuo to yield a crude solid.Recrystallize (hexane/ethyl acetate) to yield 2.67 g (57%) of the titlecompound; mp 75°-77° C.

EXAMPLE 7 1-Phenyl-3-[3-(4-ethoxybenzyloxy)phenyl]-2-propyne-1-one Stepa: 3-(4-Ethoxybenzyloxy)-2',2'-dibromostyrene

Mix 4-ethoxybenzaldehyde (3.3 g, 22 mmol), sodium borohydride (830 mg,22 mmol) and absolute ethanol (25 mL). Stir at room temperature untilthe reaction is complete, pour onto dilute hydrochloric acid and extractinto ethyl acetate. Separate the organic phase and extract the aqueousphase with ethyl acetate (2×). Combine the organic phases and dry(MgSO₄). Evaporate the solvent in vacuo and purify by silica gelchromatography to give 4-ethoxybenzyl alcohol.

Mix 4-ethoxybenzyl alcohol (3.35 g. 22 mmol), 3-hydroxybenzaldehyde(2.69 g, 22 mmol), triphenylphosphine (5.8 g, 22 mmol) anddiethylazadicarboxylate (3.83 g, 22 mmol) in tetrahydrofuran (25 mL).Stir at room temperature under anhydrous atmosphere until the reactionis done. Concentrate the solution in vacuo and dilute with a smallquantity of ethyl ether. Filter any precipitate and concentrate thefiltrate in vacuo. Purify by silica gel chromatography to give3-(4-ethoxybenzyloxy)benzaldehyde.

Mix carbon tetrabromide (10.61 g, 32 mmol) and methylene chloride (15mL), place under argon atmosphere and cool to 0° C. Add, by dropwiseaddition, a solution of triphenylphosphine (16.77 g, 64 mmol) inmethylene chloride (15 mL) and stir at 0° C. for 30 minutes. Add, bydropwise addition, 3-(4-ethoxybenzyloxy)benzaldedyde (4.10 g, 16 mmol)in methylene chloride (15 mL). Remove the ice bath and stir at roomtemperature for 1 hour. Pour onto silica gel and elute with 5% ethylacetate/hexane. Evaporate the solvent in vacuo to yield the titlecompound.

Step b: 3-(4-Ethoxybenzyloxy)phenylacetylene

Place n-butyllithium (9 mL of a 2.5M solution in hexane, 22.5 mmol) andtetrahydrofuran (50 mL) under an argon atmosphere and cool in adry-ice/acetone bath. Add a solution of3-(4-ethoxybenzyloxy)-2',2'-dibromostyrene (4.41 g, 10.7 mmol) intetrahydrofuran and stir for 1 hour at low temperature. Remove thecooling bath and stir for 1 hour at room temperature. Pour the solutioninto saturated aqueous ammonium chloride and extract into ethyl ether.Separate the organic phase, dry (MgSO₄), filter and evaporate thesolvent in vacuo to yield the title compound.

Step c: 1-Phenyl-3-[3-(4-ethoxybenzyloxy)phenyl]-2-propyne-1-one

Place lithium hexamethyldisilazane (2 mL of a 1M solution intetrahydrofuran, 2 mmol) and tetrahydrofuran (8 mL) under an argonatmosphere and cool to 0° C. Add a solution of3-(4-ethoxybenzyloxy)phenylacetylene (0.51 g, 2 mmol) in tetrahydrofuranand stir the solution for 45 minutes at 0° C. AddN-methoxy-N-methylbenzamide (0.4 g, 2.4 mmol), remove the cooling bathand stir at room temperature for 1 hour. Partition the mixture betweenethyl ether and water, separate the organic phase and dry (MgSO₄).Filter and evaporate the solvent in vacuo to yield an oil. Filterthrough silica gel and elute with 10% ethyl acetate/hexane to yield acrude solid. Purify by silica gel chromatography to give the titlecompound.

EXAMPLE 81-Phenyl-3-[3-(4-(dimethylamino)benzyloxy)phenyl]-2-propyne-1-one Stepa: 3-(4-(Dimethylamino)benzyloxy)-2',2'-dibromostyrene

Mix 4-dimethylaminobenzaldehyde (3.3 g, 22 mmol), sodium borohydride(830 mg, 22 mmol) and absolute ethanol (25 mL). Stir at room temperatureuntil the reaction is complete, pour onto dilute hydrochloric acid andextract into ethyl acetate. Separate the organic phase and extract theaqueous phase with ethyl acetate (2×). Combine the organic phases anddry (MgSO₄). Evaporate the solvent in vacuo and purify by silica gelchromatography to give 4-(dimethylamino)benzyl alcohol.

Mix 4-(dimethylamino)benzyl alcohol (3.35 g. 22 mmol),3-hydroxybenzaldehyde (2.69 g, 22 mmol), triphenylphosphine (5.8 g, 22mmol) and diethylazadicarboxylate (3.83 g, 22 mmol) in tetrahydrofuran(25 mL). Stir at room temperature under anhydrous atmosphere until thereaction is done. Concentrate the solution in vacuo and dilute with asmall quantity of ethyl ether. Filter any precipitate and concentratethe filtrat in vacuo. Purify by silica gel chromatography to give3-(4-(dimethylamino)-benzyloxy) benzaldehyde.

Mix carbon tetrabromide (10.61 g, 32 mmol) and methylenechloride (15mL), place under argon atmosphere and cool to 0° C. Add, by dropwiseaddition, a solution of triphenylphosphine (16.77 g, 64 mmol) inmethylene chloride (15 mL) and stir at 0° C. for 30 minutes. Add, bydropwise addition, 3-(4-(dimethylamino)benzyloxy)benzaldedyde (4.10 g,16 mmol) in methylene chloride (15 mL). Remove the ice bath and stir atroom temperature for 1 hour. Pour onto silica gel and elute with 5%ethyl acetate/hexane. Evaporate the solvent in vacuo to yield the titlecompound.

Step b: 3-(4-(Dimethylamino)benzyloxy)phenylacetylene

Place n-butyllithium (9 mL of a 2.5M solution in hexane, 22.5 mmol) andtetrahydrofuran (50 mL) under an argon atmosphere and cool in adry-ice/acetone bath. Add a solution of3-(4-(dimethylamino)benzyloxy)-2',2'-dibromostyrene (4.41 g, 10.7 mmol)in tetrahydrofuran and stir for 1 hour at low temperature. Remove thecooling bath and stir for 1 hour at room temperature. Pour the solutioninto saturated aqueous ammonium chloride and extract into ethyl ether.Separate the organic phase, dry (MgSO₄), filter and evaporate thesolvent in vacuo to yield the title compound.

Step c:1-Phenyl-3-[3-(4-(dimethylamino)benzyloxy)phenyl]-2-propyne-1-one

Place lithium hexamethyldisilazane (2 mL of a 1M solution intetrahydrofuran, 2 mmol) and tetrahydrofuran (8 mL) under an argonatmosphere and cool to 0° C. Add a solution of3-(4-(dimethylamino)benzyloxy)phenylacetylene (0.51 g, 2 mmol) intetrahydrofuran and stir the solution for 45 minutes at 0° C. AddN-methoxy-N-methylbenzamide (0.4 g, 2.4 mmol), remove the cooling bathand stir at room temperature for 1 hour. Partition the mixture betweenethyl ether and water, separate the organic phase and dry (MgSO₄).Filter and evaporate the solvent in vacuo to yield an oil. Filterthrough silica gel and elute with 10% ethyl acetate/hexane to yield acrude solid. Purify by silica gel chromatography to give the titlecompound.

Alternatively, compounds of formula (1) can be prepared according to theprocedure set forth in Scheme B, wherein all substituents, unlessotherwise indicated, are previously described. ##STR3##

In general, a 1-phenyl-3-phenyl-2-propyne-1-one compound of structure 5can be prepared in a 2-step process from the appropriate phenylacetylenecompound of structure 3 (Scheme A).

In step a, the appropriate α-[(phenyl)ethynyl]-benzenemethanol compoundof structure 6 can be prepared by first reacting the appropriatephenylacetylene compound of structure 3 with a non-nucleophilic base,such as n-butyllithium, lithium hexamethyldisilazane, or lithiumdiisopropylamide, in a suitable aprotic solvent, such astetrahydrofuran. The corresponding lithium acetylide can then be reactedwith the appropriate benzaldeyde compound of structure 1 to give theappropriate α-[(phenyl)ethynyl]-benzenemethanol compound of structure 6.

In step b, the appropriate α-[(phenyl)ethynyl]-benzenemethanol compoundof structure 6 can be oxidized to the corresponding1-phenyl-3-phenyl-2-propyne-1-one compound of structure 5 by techniquesand procedures well known and appreciated by one of ordinary skill inthe art. For example, the appropriateα-[(phenyl)ethynyl]-benzenemethanol compound of structure 6 can beoxidized to the corresponding 1-phenyl-3-phenyl-2-propyne-1-one compoundof structure 5 by means of either a Swern oxidation (dimethylsulfoxide,oxalyl chloride, and triethylamine), by means of pyridinium dichromateoxidation in a suitable aprotic solvent, such as methylene chloride orby means of barium manganate oxidation in a suitable aprotic solvent,such as methylene chloride.

Starting materials for use in the general synthetic procedure outlinedin Scheme B are readily available to one of ordinary skill in the art.

The following examples present typical syntheses as described in SchemeB. These examples are understood to be illustrative only and are notintended to limit the scope of the present invention in any way.

EXAMPLE 91-(4-Dimethylaminophenyl)-3-(3-benzyloxyphenyl)-2-propyne-1-one Step a:1-(4-Dimethylamino)-α-[(3-benzyloxyphenyl)ethynyl]-benzenemethanol

Place lithium hexamethyldisilazane (3 mL of a 1M solution intetrahydrofuran, 3 mmol) under argon atmosphere and cool to 0° C. Add3-benzyloxyphenylacetylene (0.62 g, 3 mmol) (see Example 6) intetrahydrofuran (20 mL) and stir for 1 hour at 0° C. Add a solution of4-dimethylaminobenzaldehyde (0.42 g, 2.8 mmol) in tetrahydrofuran.Remove the ice bath and stir at room temperature for 1 hour. Pour ontoethyl ether and water, separate the organic phase and evaporate thesolvent in vacuo. Filter through silica gel, eluting with 25% ethylacetate/hexane. Evaporate the solvent in vacuo to yield 0.58 g of thetitle compound.

Step b: 1-(4-Dimethylaminophenyl)-3-(3-benzyloxyphenyl)-2-propyne-1-one

Mix 1-(4-dimethylamino)-α-[(3-benzyloxyphenyl)ethynyl]-benzenemethanol(0.55 g, 1.5 mmol), pyridinium dichromate (0.75 g, 2 mmol) and methylenechloride (15 mL). Stir at room temperature for 4 hours. Dilute withethyl ether (100 mL) and filter the solid. Evaporate the solvent andcrystallize (hexane/ether) the residual oil. Recrystallize(hexane/chloroform) to yield 0.1 g of the title compound; mp 134°-136°C.

EXAMPLE 10 1-(3-Benzyloxyphenyl)-3-(3-benzyloxyphenyl)-2-propyne-1-oneStep a: 1-(3-Benzyloxy)-α-[(3-benzyloxyphenyl)ethynyl]-benzenemethanol

Place lithium hexamethyldisilazane (1.5 mL of a 1M solution intetrahydrofuran, 1.5 mmol) under argon atmosphere and cool to 0° C. Add3-benzyloxyphenylacetylene (0.31 g, 1.5 mmol) (see Example 6) intetrahydrofuran (10 mL) and stir for 1 hour at 0° C. Add a solution of3-benzyloxybenzaldehyde (0.32 g, 1.5 mmol) in tetrahydrofuran. Removethe ice bath and stir at room temperature for 1 hour. Pour onto ethylether and water, separate the organic phase and evaporate the solvent invacuo. Filter through silica gel, eluting with 25% ethyl acetate/hexane.Evaporate the solvent in vacuo to yield 0.44 g of the title compound.339E-175

Step b: 1-(3-Benzyloxyphenyl)-3-(3-benzyloxyphenyl)-2-propyne-1-one

Mix 1-(3-benzyloxy)-α-[(3-benzyloxyphenyl)-ethynyl]-benzenemethanol(0.44 g, 1.5 mmol), pyridinium dichromate (0.78 g, 2 mmol) and methylenechloride (15 mL). Stir at room temperature for 4 hours. Dilute withethyl ether (100 mL) and filter the solid. Evaporate the solvent andpurify by silica gel chromatography to yield 0.2 g of the titlecompound.

The following compounds can be prepared by procedures analogous to thosedescribed above in Examples 1-10:

1-Phenyl-3-(3,4,5-triethoxyphenyl)-2-propyne-1-one;

1-Phenyl-3-[4-(4-methoxybenzyloxy)phenyl]-2-propyne-1-one;

1-Phenyl-3-[(3-benzyloxy)phenyl]-2-propyne-1-one;

1-(3,4,5-triethoxyphenyl)-3-phenyl-2-propyne-1-one;

1-(4-Dimethylaminophenyl)-3-(4-benzyloxyphenyl)-2-propyne-1-one;

1-(3-Benzyloxyphenyl)-3-(4-benzyloxyphenyl)-2-propyne-1-one.

One embodiment of the present invention provides a method of inhibitingof calcium uptake in leukocytes. As used herein the term "inhibition"refers to any process whereby the uptake of calcium into leukocytes isslowed, interrupted, arrested, or stopped and does not necessarilyindicate a total elimination of calcium uptake into the affected cellsor tissues.

The terms calcium and calcium ion (Ca⁺²) may be used interchangablyherein to refer to the element of calcium and its ion states which evermay be actively involved the cellular processes of leukocytes.Furthermore, it is understood that inhibition of calcium uptake intoleukocytes by compounds of formula (1) may affect one or more ofelemental states of calcium and should not be limited to any ionic formof calcium and/or its association with any particular counterion; forexample, calcium chloride, calcium carbonate, calcium fluoride, etc. areall known and would be considered within the definition of calcium usedherein.

Calcium uptake into leukocytes is one of the signatory events ofleukocyte activation and serves as a second messenger to the cell. Oftenthe uptake of calcium is dependent on some first messenger, as forexample, the binding of a ligand to its cell surface receptor. Bindingof the ligands to their receptors can serve to open Ca⁺² channels,allowing Ca⁺² to enter the cytosol, where it then functions as a secondmessenger. When cells are activated, corresponding to an influx of Ca⁺²a number of events can occur which lead or contibute to immunological orinflammatory based diseases.

Leukocytes encompass a class of cells of the immune system that arehistologically and biochemically distinct from other cells of the immunesystem. Leukocytes as referred to herein encompass five major classes ofcells: neutrophils, basophils, eosinophils, macrophages, andlymphocytes. Within these cell classes can be further classifications ofcell types; for example, neutrophils can be further understood toinclude polymorphonuclear leukocytes. All major classes of leukocytesare related in that they are derived from a progenitor myeloid stemcell. Although the term neutrophil is used extensively herein, it isused to be exemplary of the action of leukocytes as all cells derivedfrom myeloid stem cells and is not meant as any limitation upon the useor administration of compounds of the formula (1) that may be causativeor symptomatic from the action of any particular type of leukocytes orcell of the immune system. Furthermore, the action of a leukocytes,wherein the process is to limit or stop the uptake of calcium to affecta cure in a disease process is not limited by the disease being effectedby multiple cell or tissue types and encompasses a complex interactionof cellular and tissue interactions. One embodiment of the invention isthe inhibition of calcium uptake in leukocytes wherein the effect ofinhibition results in a beneficial modulation of phagocytic functions ofleukocytes. It is therefore further understood that leukocytic cells canbe classified by the functionality inhibited; for example, one could usethe term phagocytes or cells with phagocytic functions as a group orsubgroup of cells encompassing the cells known as leukocytes herein.

Leukocytes respond to a large variety of inflammatory and immuneconditions. During these events receptor activation or general cellularactivation results in an altered cellular physiology wherein theleukocyte becomes activated. Often the term "activation" is used torefer to the process or state of leukocytes that have become activatedthat can be characterized by the cells response to take up calcium whilebecoming activated or maintaining the state of activation.

The activation of cellular oxidative and nonoxidative mechanisms thatresult from leukocyte activation play an important role in thedevelopment of a number of immune diseases processes. As part of thecellular defense system, including the adverse symptoms and tissueinjury of the host, activation of participating leukocytes is in partcalcium dependent. The noninfectious disease processes in whichleukocytes, primarily the neutrophils, are thought to play an integralpart in the symptoms and tissue injury of the host include; gout,rheumatoid arthritis, immune vasculitis, glomerulonephritis, neutrophildermatoses, inflammatory bowel disease, myocardial infarction, adultrespiratory distress syndrome, emphysema, asthma, thermal injuryassociated with hemolysis, and malignant neoplasms at sites of chronicinflammation (Malech and Gallin; 1987).

In a number of autoimmune diseases, such as gout, autoimmunearthritides, autoimmune vasculitis and some forms of glomerulonephritis,neutrophils are found to accumulate in the areas of the joint and serveto contribute to the destruction of joint and soft tissue. Although anumber of disease processes are involved in the patho-physiology ofthese diseases the recruitment of neutrophils into the area is welldocumented and may be responsible to a large part of the observablehistopathological aspects of these diseases. Therein, a number ofstudies in model systems have clearly demonstrated that nonoxidativeprocesses which would result from neutrophil activation, particularlythe release of neutrophil elastase and other proteolytic and glycolyticenzymes, can directly damage host tissues.

A large variety of dermatopathic disorders are associated with theinfiltration of neutrophils into the ectodermal and dermal tissues ofthe skin. Included with the diseases of the skin wherein neutrophilsparticipate in the progression of the disease, but not limited to, areforms of psoriasiform dermatoses, vessel-based neutrophilic dermatosesas found in Sweet's syndrome and Behcet's disease, and pyodermagangrenosum. In these disease processes neutrophils serve to augment andmaintain the inflammmatory symptoms, enhance tissue injury, and maythereby prevent healing.

A number of other diseases, such as inflammatory bowel disease andmyocardial infarction, are related to defective neutrophil functionwithin a particular organ. Studies in animals suggest that abnormalitiesin the circulation of the neutrophils in the bowel result in an abnormalactivation of these cells. In the same sense, neutrophils have beenshown to be recruited to the infarction in the myocardial tissues and toplay a part in enhancing tissue injury after a myocardial infarction.

The pathogenesis of a number of respiratory disorders including adultrespiratory distress syndrome (ARDS), emphysema, and asthma have beenassociated with neutrophil infiltration and aggravation of the disease.Neutrophil mediated oxidative damage of the pulmonary tissues may be animportant component of the pathogenesis of the these respiratorydisorders. Further, neutrophil accumulation with the release of elastasemay have an important role in the tissue destruction occurring inemphysema and may have an important role in the late phases of theinflammatory response in allergic asthma. The allergic response, duringwhich neutrophils release their constituents, may occur from the releaseof histamine by mast cells.

Neutrophil activation may serve in the general pathogenesis of diseasesrelated to complement activation. It is well known that complement canserve to activate neutrophils with the concomitant production ofhydroxyl radicals. The production of hydroxyl radicals from neutrophilactivation may therein be responsible for erythrocyte fragility andintravascular hemolysis associated with complement activation.

The term "inflammation" as used herein is considered as a process thatmost often involves leukocytes of the immune system and is the processinvolved in a "inflammatory condition". Clinically, the cardinal signsof inflammation may include one or more of the following symptoms:redness, swelling, heat, pain, loss of function, and fever. Howevercertain cellular events accompanying inflammation include, but notlimited to, leukocyte activation, leukocyte margination and emigration,and leukocyte exudation. It is also considered that any condition inwhich there exist an early or latent inflammatory state, leukocytes mayparticipate in its underlying causes or serve to perpetuate or expandthe disease state or condition. Therein, processes involving aninflammatory condition are considered herein to involve leukocytes ofthe immune system and as such can be viewed within the repertoire of"immune diseases" and conditions affected by leukocytes capable of beingtreated with compounds of the formula (1). It is also considered thatcompounds of the present invention that affect the uptake of calciuminto leukocytes are thought to be potentially beneficial in theamelioration of conditions involving inflammation and/or immunediseases.

The development of methods like those of the present invention toabrogate neutrophil responses or to inactivate neutrophil oxidativemetabolites and granule contents will be useful in limiting tissuedestruction in these noninfectious inflammatory diseases.

Platelets (thrombocytes) are non-nucleated cells of the blood that arederived from megakaryocytes of the the bone marrow. Platelets are alsorequired for the clotting of blood and help repair breaches in the wallsof blood vessels. The platelet response to form clots and to repairdamaged vascular tissue can also have a number of detrimental sideeffects; for example, during ischemic reperfusion the thrombus formationand associated platelet aggregation can lead eventually to occlusion ofthe artery. It is also well known in the field that the closing orocclusion of the walls of the arteries and veins can lead to theoccurrence of a myocardial infarction. It is also well known thatcalcium is required in the blood clotting process and for thrombusformation; for example modulation of the clotting response by EDTA andother chelators that bind calcium is firmly established in the field. Itis thereby envisioned that inhibiting calcium uptake into platelets mayrender itself as a viable means by which to modulate platelet activationtherapeutically, which would include the release of vasoactive mediatorsand formation of platelet aggregates, where such use is administered toa patient in need thereof.

As used herein, the term "patient" refers to a warm blooded animal suchas a mammal which is afflicted with a particular immune disease state.It is understood that dogs, cats, rats, mice, horses, cattle, sheep, andhumans are examples of animals within the scope of the meaning of theterm.

The compounds of formula (1) are believed to exert their inhibitoryeffect on calcium uptake in leukocytes and platelets and thereby providerelief of calcium dependent mechanisms involved in immunoregulation,including inflammatory and immune diseases. However, it is understoodthat the present invention is not limited by any particular theory orproposed mechanism to explain its effectiveness in and end-useapplication. It is also understood that the use of the term compounds ofthe formula (1) is also inclusive of all its radicals that includederivatives (1a), (1b), (1c), and (1d).

As is well known and appreciated by those skilled in the art, variousdisease states in certain inflammatory and immune diseases arecharacterized by an events that lead calcium uptake into leukocytes. Asused herein, events that lead to calcium uptake in leukocytes can eitherrefer to the initial events leading to the initial condition or eventsassociated with maintaining an inflammatory or immune condition.Furthermore, it is also well known and appreciated by those skilled inthe art that certain cardiovascular diseases are characterized by eventsleading to calcium uptake in platelets and such events may thereto refereither to the initial events leading to the condition or to eventsassociated with maintaining the cardiovascular disease state.

More specifically, the present invention provides a method for thetreatment of a patient afflicted with a calcium dependent disease statein leukocytes, wherein such disease states may be, but not limited to,an inflammatory or immune disease, that comprises the administration ofa effective calcium uptake inhibitory amount of a compound of formula(1).

A therapeutically effective calcium uptake inhibitory amount of acompound of formula (1) refers to an amount which is effective incontrolling the activation of leukocytes involved in a immune diseasestate. The term "controlling the activation" is intended to refer to allprocesses wherein there may be a slowing, interrupting, arresting, orstopping of the progression of the immune disease and does notnecessarily indicate a total elimination of all disease symptoms.

Furthermore, it is also well known and appreciated by those skilled inthe art that certain cardiovascular diseases are characterized by eventsleading to calcium uptake in platelets and such events may thereto refereither to the initial events leading to the condition or to eventsassociated with maintaining the cardiovascular disease state.

The present invention also provides a method for the treatment of apatient afflicted with a calcium dependent disease state in platelets,wherein such disease states may be, but not limited to, cardiovasculardiseases, that comprises the administration of a effective calciumuptake inhibitory amount of a compound of formula (1).

A therapeutically effective calcium uptake inhibitory amount of acompound of formula (1) refers to an amount which is effective incontrolling the platelet function involved in a cardiovascular diseasestate. The term controlling the platelet function involved in acardiovascular disease refers to slowing, interrupting, arresting, orstopping the progression of the cardiovascular disease and does notnecessarily indicate a total elimination of all disease symptoms.

A therapeutically effective inhibitory amount in either a immune orcardiovascular condition or disease, can be readily determined by theattending diagnostician, as one skilled in the art, by the use ofconventional techniques and by observing results obtained underanalogous circumstances. In determining the therapeutically effectivedose, a number of factors are considered by the attending diagnostician,including, but not limited to: the species of mammal; its size, age, andgeneral health; the specific disease involved; the degree of orinvolvement or the severity of the disease; the response of theindividual patient; the particular compound administered; the mode ofadministration; the bioavailability characteristic of the preparationadministered; the dose regimen selected; the use of concomitantmedication; and other relevant circumstances.

A therapeutically effective amount of a compound of formula (1) isexpected to vary from about 0.1 milligram per kilogram of body weightper day (mg/kg/day) to about 100 mg/kg/day. Preferred amounts areexpected to vary from about 0.5 to about 10 mg/kg/day.

In effecting treatment of a patient afflicted with a disease statedescribed above, a compound of formula (1) can be administered in anyform or mode which makes the compound bioavailable in effective amounts,including oral and parenteral routes. For example, compounds of formulacan be administered orally, subcutaneously, intramuscularly,intravenously, transdermally, intranasally, rectally, topically, and thelike. Oral administration is generally preferred. One skilled in the artof preparing formulations can readily select the proper form and mode ofadministration depending upon the particular characteristics of thecompound selected the disease state to be treated, the stage of thedisease, and other relevant circumstances.

The compounds can be administered alone or in the form of apharmaceutical composition in combination with pharmaceuticallyacceptable carriers or excipients, the proportion and nature of whichare determined by the solubility and chemical properties of the compoundselected, the chosen route of administration, and standardpharmaceutical practice. The compounds of the invention, while effectivethemselves, may be formulated and administered in the form of theirpharmaceutically acceptable acid addition salts for purposes ofstability, convenience of crystallization, increased solubility and thelike.

In another embodiment, the present invention provides pharmaceuticalcompositions comprising a effective amount of a compound of formula (1)in admixture or otherwise in association with one or morepharmaceutically acceptable carriers or excipients. The term "effectivecalcium uptake inhibitory amount" as applied to compounds of formula (1)refers to effective inhibitory amounts as appropriate to inhibit theuptake of calcium in leukocytes wherein such inhibition may be for apatient suffering from an immune or inflammatory disease. Further theterm "effective calcium uptake inhibitory amount" as applied tocompounds of (1) also refers to effective inhibitory amounts asappropriate to inhibit the uptake of calcium in platelets wherein suchinhibition may be for a patient suffering from a disease associated withthe cardiovascular system.

The pharmaceutical compositions are prepared in a manner well known inthe pharmaceutical art. The carrier or excipient may be a solid,semi-solid, or liquid material which can serve as a vehicle or mediumfor the active ingredient. Suitable carriers or excipients are wellknown in the art. The pharmaceutical composition may be adapted fororal, parenteral, or topical use and may be administered to the patientin the form of tablets, capsules, suppositories, solution, suspensions,or the like.

The compounds of the present invention may be administered orally, forexample, with an inert diluent or with an edible carrier. They may beenclosed in gelatin capsules or compressed into tablets. For the purposeof oral therapeutic administration, the compounds may be incorporatedwith excipients and used in the form of tablets, troches, capsules,elixirs, suspensions, syrups, wafers, chewing gums and the like. Thesepreparations should contain at least 4% of the compound of theinvention, the active ingredient, but may be varied depending upon theparticular form and may conveniently be between 4% to about 70% of theweight of the unit. The amount of the compound present in compositionsis such that a suitable dosage will be obtained. Preferred compositionsand preparations according to the present invention are prepared so thatan oral dosage unit form contains between 5.0-300 milligrams of acompound of the invention.

The tablets, pills, capsules, troches and the like may also contain oneor more of the following adjuvants: binders such as microcrystallinecellulose, gum tragacanth or gelatin; excipients such as starch orlactose, disintegrating agents such as alginic acid, Primogel, cornstarch and the like; lubricants such as magnesium stearate or Sterotex;glidants such as colloidal silicon dioxide; and sweetening agents suchas sucrose or saccharin may be added or a flavoring agent such aspeppermint, methyl salicylate or orange flavoring. When the dosage unitform is a capsule, it may contain, in addition to materials of the abovetype, a liquid carrier such as polyethylene glycol or a fatty oil. Otherdosage unit forms may contain other various materials which modify thephysical form of the dosage unit, for example, as coatings. Thus,tablets or pills may be coated with sugar, shellac, or other entericcoating agents. A syrup may contain, in addition to the presentcompounds, sucrose as a sweetening agent and certain preservatives, dyesand colorings and flavors. Materials used in preparing these variouscompositions should be pharmaceutically pure and non-toxic in theamounts used.

For the purpose of parenteral therapeutic administration, the compoundsof the present invention may be incorporated into a solution orsuspension. These preparations should contain at least 0.1% of acompound of the invention, but may be varied to be between 0.1 and about50% of the weight thereof. The amount of the inventive compound presentin such compositions is such that a suitable dosage will be obtained.Preferred compositions and preparations according to the presentinvention are prepared so that a parenteral dosage unit contains between5.0 to 100 milligrams of the compound of the invention.

The compounds of formula (1) of this invention may also be administeredtopically, and when done so the carrier may suitably comprise asolution, ointment or gel base. The base, for example, may comprise oneor more of the following: petrolatum, lanolin, polyethylene glycols, beewax, mineral oil, diluents such as water and alcohol, and emulsifiersand stabilizers. Topical formulations may contain a concentration of theformula 1 or its pharmaceutical salt from about 0.1 to about 10% w/v(weight per unit volume).

The solutions or suspensions may also include the one or more of thefollowing adjuvants: sterile diluents such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl paraben; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylene diaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. Theparenteral preparation can be enclosed in ampules, disposable syringesor multiple dose vials made of glass or plastic.

It is understood herein that the following terms and (abbreviations)used herein are meant to be synonymous with each other:polymorphonuclear leukocytes (PMNL),[4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES),multiplicative factor of gravity used to express the centrifical forceexerted during centrifugation (x g), milliliters (mL), degreescentigrade (°C.), calcium ion (Ca⁺²), nanometers (nm), dimethylsulfoxide(DMSO), ethylenebis (oxyethylenenitrilo)-tetraacetic acid (EGTA) grams(g), milligrams (mg), nanograms (ng), molar (M), millimolar (mM),micromolar (uM), opsonized zymozan (OZ), phorbol myristate acetate(PMA), formyl-methionyl-leucylphenylalanine (fMLP), leukotriene B₄(LTB₄).

Compounds are often represented by a three letter abreviation "MDL" aspace and a five or six digit extension. Therein it is understood thefollowing representations: 1-phenyl-3-[3-(4 methoxybenzyloxy)phenyl]-2-propyne-1-one (MDL 100,225);1-phenyl-3-(4-pyridinyl)-2-propyne-1-one (MDL 101,098);1-(4-dimethylaminophenyl)-3-(4-pyridinyl)-2-propyne-1-one (MDL 102,175);1-phenyl-3-[(4-benzyloxy)phenyl]-2-propyne-1-one (MDL 101,097);1-(4-dimethylaminophenyl)-3-(4-pyridinyl)-2-propene-1-one (MDL 101,240);1-phenyl-3-(4-pyridinyl)-2-propene-1-one (MDL 29,355);1-(4-diethylaminophenyl)-3-(4-quinolinyl)-2-propyne-1-one (MDL 102,387).

The response of leukocytes to activation signals (stimulants) can beassessed by in vitro measurement of intracellular calcium or bymeasurement of released factors, such as superoxide anion, andmyeloperoxidase. Further the response of leukocytes to stimulation invivo can be assessed in animal models of edema, such as the mustard oilinduced mouse ear edema model and the carrageenin induced rat paw edemamodel. Inventors of the present subject matter therein demonstrate themethod of using the claimed compounds and their utility in the precedingassays.

Materials

Sprague-Dawley rats (150-250 grams) were from Harlan Sprague-Dawley(Indianapolis, Ind.). Hanks' balanced salts (HBSS; Gibco, Grand Island,N.Y.) was supplemented with 20 mM HEPES and adjusted to pH 7.4. Sodiumcaseinate was from ICN Biochemical (Cleveland, Ohio). Fura-2/AM was fromMolecular Probes (Eugene, Oreg.). Ionomycin was from Cal Biochem. (SanDiego, Calif.). Superoxide dismutase was from DDI Pharmaceuticals(Mountain View, Calif.). All other reagents were from Sigma Chemical Co.(St. Louis, Mo.). All purchased reagents were used as received.

Isolation of Polymorphonuclear leukocytes (PMNL)

Rat PMNL were obtained from exudates resulting from peritoneal injectionof 6 mL of 8% sodium caseinate. Eighteen hours after injection rats werekilled by carbon dioxide inhalation and the peritoneal cavity lavagedwith Hank's balanced salt solution (HBSS). After concentration of thePMNL by centrifugation, contaminating erythrocytes were removed byhypotonic lysis. The PMNL were then washed twice by centrifugation(400×g for 10 minutes) and resuspended in HBSS. Cell viability wasgreater than 90% by Trypan Blue exclusion and greater than 90% of thecells were PMNL by Wright-Giemsa stain.

Measurement of Intracellular Calcium

Intracellular calcium levels were obtained using Fura-2 as fluorescentindicator (Grynkiewicz et al., 1985). Fura-2 was loaded into PMNL as theacetoxymethyl ester (Fura-2/AM) using the method of Korchak et al.,(1988). Specifically, 1×10⁸ PMNL/mL were incubated in HBSS with 10 μMFura-2/AM for 10 minutes at 37° C. The cells suspension was then dilutedten fold with HBSS and further incubated for another 20 minutes. Thecells were then centrifuged (400×g for 8 minutes at 25° C.) and the PMNL(1×10⁷ cells/mL) resuspended in HBSS containing 1% bovine serum albumin.Cells were stored at room temperature and used within 3 hours.

Intracellular calcium level determinations were made using a dualwavelength spectrofluorometer (Photon Technology International, NewBrunswick, N.J.). Two mL of PMNL suspension (1×10⁷ cells/mL) werestirred magnetically at 37° C. Cells were preincubated with thecompounds for 15 minutes at 37° C. prior to activation. Where compoundswere dissolved in DMSO and added directly, the DMSO never exceeded 0.3%in the final cell suspension. Total change in volume with addition ofcompounds or vehicle and stimuli never exceeded 3%. Intracellularcalcium concentrations were calibrated by setting a maximum calciumlevel in the cells with the addition of 7 uM ionomycin to PMNL in HBSScontaining 1% BSA. This was followed by treatment with 20 mM EGTA for 1hour to obtain a minimum calcium level. The method of Grynkiewiez et al.(1985) was used to quantitate intracellular calcium levels, assumingK_(D) for Fura-2 to be 240 nM. Each experiment was performed andrepeated on three separate occasions and the pattern of calcium changesare representative of these experiments.

Myeloperoxidase (MPO) Release

MPO release was determined using the MPO catalyzed oxidation ofo-dianisidine. The experiments were carried out in 96-well microtiterplates (Webster and Henson, 1978). In each well, 50 microliter (μl)aliquots of a PMNL suspension (2×10⁷ cell/mL in HBSS) were incubatedwith 50 μl of HBSS containing vehicle (0.3% DMSO) or inhibitor for 30minutes at 37° C. The final DMSO concentration never exceeded 0.3% andwas constant throughout each experiment. To activate the PMNL, 50 μl ofone of the following were added for 30 minutes at 37° C.:N-formyl-Met-Leu-Phe (fMLP; 0.1 μM); phorbol myristate acetate (PMA, 200ng/mL); or rat serum opsonized zymosan (OZ; 2.6 mg/mL, prepared by themethod of Ward et al., 1983). The stimuli were added in 50 μl HBSS whichalso contained the compounds or vehicle, as appropriate, to avoidchanging the inhibitor concentration. Cell activation was terminated bycentrifugation of the plates at 600×g for 5 minutes at 22° C. Aliquots(100 μl) of supernatant were removed from each well and transferred to a96-well flatbottomed microtiter plate for the MPO assay. To each aliquotof supernatant was added 100 μl of a freshly prepared solutioncontaining 50 μl of 0.2M sodium phosphate buffer (pH 6.2), 25 μl of 3.9mM O-dianisidine and 25 μl of 0.015M hydrogen peroxide. Blanks wereobtained using the same solution without hydrogen peroxide. The plateswere mixed and then incubated at room temperature in the dark for about15 minutes. Absorbances of the samples were measured at 450 nm.

Superoxide Anion (SOA) Production

Superoxide anion generation was measured using a method comparable tothat of Leslie (1987) using microtiter plates, and carried out inpresence of catalase (Arthur et al., 1987). Aliquots (50 μl) of a ratPMNL suspension (2×10⁷ cells/mL in HBSS) were incubated with 50 μl ofHBSS containing vehicle (0.3% DMSO) or compound for 30 minutes at 37° C.as above. One hundred microliters of buffer containing 5.98 mg/mL ofcytochrome C (Sigma Type III) and 0.1 mg/mL catalase, plus the compoundswhere appropriate, were then added to each sample. A blank forspectrophotometric measurement, containing unstimulated cells in thepresence of cytochrome C, catalase and SOD, was also prepared. Toactivate the PMNL, 75 μl of PMA or OZ were added to achieve the finalconcentration of cell activator (plus vehicle or inhibitor as describedabove for MPO release) and the cells further incubated at 37° C. for 60minutes. The activation was terminated by centrifugation at 600×g for 5minutes at 4° C. Aliquots (200 μl) of supernatant were then transferredto a second, flat-well microtiter plate and measuredspectrophotometrically at 550 nm.

EXAMPLE 11 Effect of Various Stimuli on PMNL Intracellular Calcium

Rat PMNL preloaded with Fura-2 were stimulated with fMLP and levels ofintracellular calcium were followed as shown in FIG. 1. The chemotacticpeptide fMLP produced a biphasic response with a first peak reaching amaximum after 30 seconds and a second peak maximizing around 100 secondsafter giving the peptide stimulus.

The biphasic response is consistent with those seen by Korchak et al.,(1986) for human neutrophils wherein the peak of the responsecorresponds to an early release of intracellular calcium, as would beproduced from a response with LTB₄. The second phase of the response,corresponding to the second peak at about 100 seconds after stimulation,is associated with an influx of extracellular calcium, typical from aConcanavalin A induced response. The biphasic response is interpreted bythose skilled in the art to mean that the first phase is the release ofintracellular calcium and the second phase corresponds to the influx ofextracellular calcium.

Preincubation of PMNL with MDL 101,097 shown in FIG. 1 resulted in theselective concentration-dependent suppression of the second wave ofintracellular calcium changes. The inhibition of the second peak of theresponse by compounds of formula (1), as represented by the activitiesof MDL 101,097, can be seen to effective inhibit the calcium uptake ofthe treated PMNL. At high concentrations (>100 μm), however, compoundsdid begin to affect the first phase (data not shown). The controlresponse, whereno compounds of formula (1) are included in the fMLPstimulus, shows the observed biphasic response.

EXAMPLE 12 Effect of Compounds on Rat PMNL Enzyme Release and SuperoxideAnion Generation

Rat PMNL were isolated in HBSS containing Ca⁺². As summarized in thetable below, fMLP stimulation of rat PMNL in the presence ofcytochalasin B or with opsonized zymosan or with ionomycin results inthe release of myeloperoxidase (fMLP/MPO, OZ/MPO, and ION/MPO; columns2, 4, and 6 respectively). Addition of MDL 102,175 shows a dosedependent inhibition of both responses at the given concentrations.Similarly, PMA and OZ results in superoxide anion production which isinhibited in a dose dependent fashion by the addition of MDL 101,098(PMA/SOA and OZ/SOA; columns 8 and 10 respectively). These data are alsoconsistent with the affect that one would see if rat PMNL were deprivedof extracellular calcium (data not shown).

    __________________________________________________________________________    EFFECT OF STIMULI ON MDL 101,098                                              INHIBITION OF RAT PMN MPO AND SOA                                             CONC (uM)                                                                             2       3   4      5   6      7   8      9   10    11                 MDL 101,098                                                                           fMLP/MPO                                                                              SEM OZ/MPO SEM ION/MPO                                                                              SEM PMA/SOA                                                                              SEM OZ/SOA                                                                              SEM                __________________________________________________________________________    100     96.2    1.8 97.2   4.5 88.4   4.1 97.2   2.3 115.0 10.1               30      80.8    9.0 92.2   1.1 12.7   2.7 70.0   10.3                                                                              61.5  8.3                10      34.9    5.7 48.7   8.6 5.7    2.5 29.1   5.8 20.3  5.0                 3       7.5    6.1 14.7   7.7 0.0    1.3 -4.3   10.6                                                                              -7.5  6.6                 1       2.9    4.4  5.4   10.9                                                                              -2.8   0.7 -20.7  7.9 -18.0 3.9                __________________________________________________________________________     SEM is the standard error of the mean for the preceding numbered              experiment.                                                              

EXAMPLE 13 Mustard Oil-Induced Mouse Ear Edema

Charles River male CD-1 mice weighing 30-60 grams were divided intorandomized groups. They were dosed i.p. for 30 minutes prior or orally 1hour prior to application of 20 ul of mustard oil (10% isothiocyanate inacetone) to the right ear. After 30 minutes the mice were sacrificed andan 8 millimeter diameter circular punch biopsy of each ear was obtainedand weighed. The difference between the left (control) and right(treated) ear weights were expressed as percent increase. Results of theMustard Oil Induced Mouse Ear Edema Test are shown in the table below.

    ______________________________________                                        MUSTARD OIL INDUCED MOUSE EAR EDEMA                                           COMPOUND  DOSE (mg/Kg)  Route    % Inhibition                                 ______________________________________                                        MDL 100,225                                                                             100           i.p.     60.9 ± 11.7                                         30                     33.4 ± 14.6                                         10                     25.3 ± 17.8                               ______________________________________                                    

EXAMPLE 14 Carrageenin-Induced Rat Paw Edema

Charles River male Sprague Dawley rats weighing 90 to 100 grams wereused. Inflammation of the left hind paw was induced by a 1% carrageeninsolution (0.05 cc), injected into the plantar surface of the paw. Thecontralateral paw was injected with an equal volume of saline. Drugswere administered 75 mg/Kg i.p. (1 cc/100 grams) one hour prior to pawinjection. Three hours following the carrageenin injection thedifference in volume of the control and inflamed paw was measured. Eachpaw was immersed in a well of mercury and the volume of mercurydisplaced was recorded. Paws were uniformly dipped into the mercury upto the beginning of the hairline. A venous pressure transducer, coupledto an chart recorder, was employed to measure the small volumes ofmercury displaced. Each group measurement consists of the average offour rats, each with three separate measurements. Results of theCarrageenin Induced Rat Paw Edema test are shown in the table below.

    ______________________________________                                        CARRAGEENIN INDUCED RAT PAW EDEMA                                             Compounds      Route   % Inhibition                                           ______________________________________                                        MDL 100,225    i.p.    24.3% ± 10.3%                                       ______________________________________                                    

What is claimed is:
 1. A compound selected from formula 1: ##STR4##wherein R₁, R₂ may each time independently be:hydrogen, --N(Y₁)(Y₂),wherein Y₁ is hydrogen or C₁ -C₆ alkyl, and Y₂ is a C₁ -C₆ alkyl, orX_(z) --(Ar)--(CH₂)_(n) --O--, wherein Ar is phenyl or napthyl, n=0 or1, X_(z) =hydrogen, C₁ -C₆ alkoxy or --N(Y₁)(Y₂), wherein Y₁ and Y₂ areas previously defined and Z=0, 1, 2; with the provision that R₁, and R₂,may not simultaneously be hydrogen; and R₃ is each time independentlyhydrogen, halogen, C₁ -C₆ alkyl, or C₁ -C₆ alkoxy; or the compounds offormula 1 may be a pharmaceutically acceptable salt thereof.
 2. Acompound of claim 1 wherein the compound is1-phenyl-3-(3,4,5-trimethoxyp[r]henyl)-2-propyne-1-one.
 3. A compound ofthe structure 1-phenyl-3-[3-methoxybenzyloxy)phenyl]-2-propyne-1-one. 4.A compound of claim 1 wherein the compound is1-phenyl-3-[(4-benzyloxy)phenyl]-2-propyne-1-one.
 5. A compound of thestructure 1-(3,4,5,-trimethoxyphenyl)-3-phenyl-2-propyne-1-one.
 6. Acompound of claim 1 wherein the compound is1-phenyl-3-(3-benzyloxyphenyl)-2-propyne-1-one.
 7. A compound of claim 1wherein the compound is1-(4-dimethylaminophenyl)-3-(3-benzyloxyphenyl)-2-propyne-1-one.
 8. Acompound of claim 1 wherein the compound is1-(3-benzyloxyphenyl)-3-(3-benzyloxyphenyl)-2-propyne-1-one.
 9. Acompound of the structure1-phenyl-3-[4-(ethoxybenzyloxy)phenyl]-2-propyne-1-one.
 10. A compoundof claim 1 wherein the compound is1-phenyl-[3-(4-dimethylamino)benzyloxy)phenyl]-2-propyne-1-one.